Compositions and methods for forming and strengthening bone

ABSTRACT

Compositions are provided which stimulate bone growth. Also provided are methods for utilizing the compositions for filling in bone defects, promoting rapid fusion of bone fractures, grafts, and bone-prostheses, and promoting strengthening of osteoporotic bones.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/581,467, filed Oct. 19, 2009, now U.S. Pat. No. 8,563,040, which is acontinuation of U.S. application Ser. No. 10/071,490, filed Feb. 7,2002, now abandoned, which claims priority from U.S. application Ser.No. 09/606,768, filed Jun. 29, 2000, now U.S. Pat. No. 6,372,257, whichclaims the benefit of U.S. provisional application Ser. No. 60/141,386filed on Jun. 29, 1999, and claims the benefit of U.S. application Ser.No. 09/377,283 filed Mar. 30, 1999, now U.S. Pat. No. 6,413,278, whichclaims the benefit of and is a complete application based upon U.S.Provisional application Ser. No. 60/135,095, filed Mar. 30, 1998, whichwas converted from a non-provisional application Ser. No. 09/050,498filed Mar. 30, 1998, now abandoned. All of these applications are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to methods and compositionswhich promote the controlled and programmable growth and strengtheningof bone.

2. Description of Related Art

Various bone diseases, injuries, or surgical interventions in humans andother vertebrates result in bone defects or fractures. Bone growth orrestitution is often desired to alleviate these conditions. The bonegrowth may be desired in areas where bone previously existed and ispartially or completely absent, or where its continuity has beendisrupted. Situations where such regeneration of bone is necessary ordesirable include the healing of fractures, or increasing the bone massin osteoporotic bones. Bone growth may also be desired in areas wherebone did not previously exist. Such bone generation is desirable, e.g.,for filling defects, such as caused by removal of tumors orintervertebral discs, for correcting congenital deformities such ascleft palates, or for forming a strong connection between a prosthesissuch as a joint replacement and an adjacent bone.

Various compositions are known which are designed to encourage bonegrowth. These compositions are generally applied to bone defects orfractures to provide an osteoinductive and osteoconductive environment.Examples include those disclosed in U.S. Pat. Nos. 5,563,124; 4,642,120;5,755,792; 5,830,493; and 5,711,957; PCT Patent Publications WO94/15653; WO 95/13767; WO 98/56433; and WO 97/32591; and European PatentEP 754,466. Additionally, such compositions are available commercially,including demineralized bone matrix compositions such as Grafton®(Osteotech, Eatontown, N.J.). These compositions generally comprise aporous solid, semisolid, paste or gel material including materials suchas gelatin, hyaluronic acid, collagen, amylopectin, demineralized bonematrix, and/or calcium carbonate, to create an osteoconductiveenvironment. The compositions also often include osteoinductive growthfactors such as transforming growth factor-13, bone morphogenic protein,or basic fibroblast growth factor. It may be noted that combinations ofchitosan with demineralized bone matrix and/or ground or chippedcancellous bone are unknown. Methods for filling bone defects utilizingcompositions constituted solely of chitin or chitosan are also unknown.

In spite of the availability of numerous compositions to encourage bonegrowth, problems still occur in attempts to achieve satisfactory growthof bone to fill bone defects, connect prostheses to existing bone, andfuse fractures and bone grafts to existing bone. Therefore, there is anongoing need for new compositions to provide alternatives to existingcompositions for encouraging bone growth.

SUMMARY OF THE INVENTION

Among the several objects of the present invention may be noted theprovision of compositions which promote bone formation and growth. Amore specific object of the invention is the provision of compositionswhich stimulate bone formation and growth through the stimulation ofangiogenesis and osteogenesis. Another object of the invention is theprovision of methods for stimulating bone formation and growth throughthe use of angiogenesis-stimulating compositions.

Briefly, therefore, the present invention is directed to a compositioncomprising the following components: (a) one or more materials selectedfrom the group consisting of fibroblast growth factors, vascularendothelial growth factors, endothelial cell growth factors,transforming growth factors, chitosan, bone, platelet derivedendothelial growth factors, placental growth factors, angiogenin,interleukin-8, granulocyte colony-stimulating growth factor, andsupernatant fluid from a culture of cells known to produce angiogenicfactors; (b) a material comprising demineralized bone matrix,non-decalcified bone matrix, with or without hyaluronic acid; (c) ascaffolding material selected from the group consisting of cancellousbone, chitosan, chitosan-protein, and chitin-protein fibers; and (d) agel material selected from the group consisting of chitosan, imidazolylchitosan, methylpyrrolidinone chitosan, carbodiimide chitosan,glutaraldehyde chitosan, alginate, a mixture of alginate with chitosanor a chitosan derivative, hyaluronic acid, and a mixture of hyaluronicacid with chitosan or a chitosan derivative.

Additionally, the present invention is directed to a compositioncomprising the following components: (a) one or moreangiogenesis-stimulating materials; (b) an osteoinductive material; (c)a scaffolding material; and (d) a gel material.

Moreover, the present invention is directed to a composition comprisingthe following components: (a) one or more angiogenesis-stimulatingmaterials selected from the group consisting of fibroblast growthfactors, vascular endothelial growth factors, endothelial cell growthfactors, transforming growth factors, chitosan, bone, platelet derivedendothelial growth factors, placental growth factors, angiogenin,interleukin-8, granulocyte colony-stimulating growth factor, andsupernatant from cells known to produce angiogenic factors; (b) anosteoinductive material comprising demineralized bone matrix,non-decalcified bone matrix, with or without hyaluronic acid; (c) ascaffolding material selected from the group consisting of cancellousbone, chitosan, chitosan-protein, and chitin-protein fibers; and (d) agel material selected from the group consisting of chitosan, imidazolylchitosan, methylpyrrolidinone chitosan, carbodiimide chitosan,glutaraldehyde chitosan, alginate, and a mixture of alginate withchitosan or a chitosan derivative.

Furthermore, the present invention is directed to a composition forpromoting the growth and strengthening of bone comprising a mixture ofchitosan or chitosan derivative, cancellous bone, and demineralized bonematrix. The present invention is also directed to a composition forpromoting growth and strengthening of bone comprising a mixture ofalginate, calcium, cancellous bone, and demineralized bone matrix. Also,the present invention is directed to a composition for promoting growthand strengthening of bone comprising a mixture of chitosan or chitosanderivative, alginate, cancellous bone, and demineralized bone matrix.Additionally, the present invention is directed to a composition forpromoting growth and strengthening of bone comprising a mixture ofhyaluronic acid, cancellous bone, and demineralized bone matrix.

The present invention is also directed to a method of inducing boneformation in a vertebrate. The method comprises applying thecompositions described above to a site in the vertebrate where boneformation is desired.

In an additional embodiment, the present invention is directed to amethod of filling a bone defect. The method comprises filling the bonedefect with a rigid material consisting essentially of chitin orchitosan.

DESCRIPTION OF THE FIGURES

FIG. 1A shows the surgical anterolateral approach to the athymic nuderat models used for study of the bone allograft compositions of thisinvention as discussed in Example 1.

FIG. 1B shows the exposed femur of an athymic nude rat prior to anycreation of bone defects in the animal for the study of the boneallograft compositions of this invention as discussed in Example 1.

FIG. 1C shows the placement of a fixator plate on the exposed femur ofan athymic nude rat prior to the creation of a bone defect in the femurfor the study of the bone allograft compositions of this invention asdiscussed in Example 1.

FIG. 2A shows the creation of a bone defect in the exposed femur of anathymic nude rat after placement of the fixator plate in order to studythe bone allograft compositions of this invention as discussed inExample 1.

FIG. 2B shows the exposed femur of an athymic nude rat after theimplantation of bone allograft material of the present inventionsubsequent to placement of a fixator plate and the creation of a bonedefect in the femur of the rat as discussed in Example 1.

FIG. 3A shows the appearance of bone allograft material in the excipientof alginate without the addition of calcium after implantation of thecomposition as discussed in Example 1. The image is taken one week afterthe implantation.

FIG. 3B shows the appearance of bone allograft material in the excipientof alginate with the immediate addition of calcium after implantation ofthe composition as discussed in Example 1. The image is taken one weekafter the implantation.

FIG. 4A shows the negative controls for the study of the bone allograftcompositions of this invention as discussed in Example 1. FIG. 4A showsthe unfilled bone defect of the femur of an athymic nude rat at 12 weekspost defect formation.

FIG. 4B shows the negative controls for the study of the bone allograftcompositions of this invention as discussed in Example 1. FIG. 4B showsthe bone defect of the femur of an athymic nude rat filled with alginatecarrier alone at 12 weeks post defect implantation of alginate carrier.

FIG. 5 shows the formation of new bone 12 weeks after the implantationof a bone allograft of the current invention into the femur of anathymic nude rat with a bone defect. The bone allograft composition usedfor the rat of FIG. 5 is a mixture of demineralized bone matrix (DBM),Hyaluronic acid (HA) and purified vascular endothelial growth factor(VEGF).

FIG. 6 shows the histologic evaluation of the new bone formation of theathymic nude rat of FIG. 5.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “cancellous bone” refers to the medullaryportion of bone, devoid of hematogenous and other cellular material.Cancellous bone is generally derived from human or animal cadavers.

The term “demineralized bone matrix” (“DBM”) refers to ground corticalbone which has been demineralized. Demineralization is generallyachieved by treating the ground bone with acid, usually 0.6Nhydrochloric acid. The cortical bone may be obtained from any source,including human or animal cadavers. DBM is known to containosteoinductive growth factors.

The term “non-decalcified bone matrix” (“NBM”) refers to ground corticalbone which has not been demineralized. NBM is known to containosteoinductive growth factors.

The term “osteoconductive” refers to materials which provide aenvironment for ingrowth and orientation of osteogenic cells fromsurrounding tissues. Such materials are generally porous materials,i.e., providing latticework structures similar to cancellous bone.

The term “osteogenic” refers to the process of forming new bone. Thisformation requires signaling, modulating, and transforming molecules.

The term “osteoinductive” refers to the ability of a material to inducethe production of osteoblasts from precursor cells, in particularmesenchymal stem cells. The osteoinductive material may act directly asa growth factor which interacts with precursor cells to induce theosteoblast differentiation, or the material may act indirectly byinducing the production of osteoinductive growth factors. This inductionalso requires signaling, modulating, and transforming molecules.

When used herein, the term “biocompatible” refers to materials which,when incorporated into the invention composition, have low toxicity,acceptable foreign body reactions in the living body, and affinity withliving tissues.

“Chitin” is poly(1,4)2-amino-2-deoxy-β-D-glucan. It is an abundantpolysaccharide which forms, e.g., the structural component of the cellwalls of many fungi and the shells of insects and shellfish.

“Chitosan” is N-deacetylated chitin. Deacetylation is generallyaccomplished by treatment of chitin with alkali such as sodiumhydroxide. The degree of N-deacetylation can be controlled bycontrolling the amount of alkali treatment and time of exposure. Thelength of the chitosan polysaccharide can also be decreased by degradingthe high molecular weight molecule with, e.g., 1 N hydrochloric acid, orby enzymatic treatment. Chitosan in acidic solutions forms gels atconcentrations as low as 1% (w/w in 1% v/v acetic acid) (Bodmeier etal., 1989, Drug Devel. & Ind. Pharmacy 15:1475) and is insoluble atpH>6.5. Chitin and chitosan can be cross-linked with charged radicals(i.e. glutaraldehyde, carbodiimide, lysine, vinyl, etc.) to obtainstrongly bonded polymers that can be formed into solid or tubularstructures. For information on various other characteristics and usefulmanipulations of chitosan, see, e.g., PCT patent publication WO98/22114, which is herein incorporated by reference.

No particular form of chitin or chitosan is to be regarded asnecessarily more useful than any other form for use in the presentinvention. The skilled artisan would be able to discern without undueexperimentation whether a particular form of chitosan is useful for theparticular composition desired.

“Alginate” is a copolymer of (1,4)-α-L-guluronic acid and β-D-mannuronicacid produced by brown algae. A gel is formed when a 2% alginatesolution interacts with divalent cations such as calcium. See Gaserod etal., 1998, Biomaterials 19:1815 for additional information oncharacteristics of alginate.

The term “1 to n”, when used with the name of a growth factor, meansthat various forms of the growth factor, both known and not yetdiscovered, are intended to be included.

The contents of each of the references cited herein are hereinincorporated by reference.

The present invention provides compositions and methods which stimulatebone formation and growth in a vertebrate. These methods areparticularly useful for stimulating bone formation in humans and othermammals. The compositions are designed utilizing the unappreciatedconcept that angiogenesis is a pivotal and crucial factor for inducingbone formation and growth. Thus, angiogenesis-stimulating factors suchas vascular endothelial growth factors, platelet derived endothelialcell growth factors, basic fibroblast growth factors, and interleukin-8,or materials which are known to stimulate the production ofangiogenesis-stimulating factors (e.g., chitosan) are components whichare as important as osteoinductive and osteoconductive factors in thecompositions of the present invention.

The compositions of the invention comprise these components: a source ofan angiogenesis-stimulating agent, a source of an osteoinductive agent,an osteoconductive scaffolding component, a substance for allowing easyflow and volumetric conformation and for holding theangiogenesis-stimulating agent and osteoinductive agent and allowing theslow release of these components, and a substance for preventing thecomposition from moving away from the location where placed. It is to benoted that some materials which may be used in the compositions of thepresent invention may serve as more than one of the above components.Also, the materials of this invention may stimulate bone growth andformation by other or additional mechanisms. The invention compositionsmay also include materials which are not yet known, but which providecharacteristics relating to these components which are similar to thematerials described herein.

When a composition of the present invention is placed in a locationwhere bone formation and/or growth is desired, the components of thecomposition work together to achieve the desired purpose. Theangiogenesis-stimulating agent stimulates the growth of neovasculaturefrom existing host tissue, which is vital for the process of new boneformation. The osteoinductive component induces the formation ofbone-forming osteoblasts from precursor cells, in particular mesenchymalcells. The material holding and allowing slow release of the angiogenicand osteogenic components assures a release of these growth factors fromthe composition over a substantial period of the bone forming and growthprocesses. Where desired, this material will impart characteristics ofthe composition which allows easy flow, permitting the composition tovolumetrically fill the desired space and configuration to deliver thegrowth factors to their site of action. This filling action also servesto interfere with the ingrowth of fibrous scar-forming tissue and othertissues which could create defects, voids, etc. that may interfere withproper bone formation. The bone which does form around such ingrowntissue may be weak, permitting fractures and failures. The materialpreventing movement of the composition assures that the composition doesnot move outside of the intended site. Such movement could reduce theeffectiveness of the composition and could be injurious at a site wherebone growth is not wanted.

Any known angiogenesis-stimulating agent is useful in the compositionsof the present invention. Examples of such agents are individual growthfactors known to induce angiogenesis, such as various fibroblast growthfactors, various vascular endothelial growth factors, endothelial cellgrowth factor, various transforming growth factors, platelet derivedendothelial cell growth factor, placental growth factor, angiogenin,interleukin-8, or granulocyte colony-stimulating growth factor. Thesegrowth factors may be provided to the composition in purified form inconcentrations ranging from about 10⁻⁶ to 30 mg/ml, or in equivalentconcentrations in an impure or incompletely characterized form, such asin bone, and in cells known to produce angiogenic factors such asendothelial cells, WI-38 cells (human embryonic lung cells), U937 cells(human lymphoma cells), or SK-Hep (human hepatoma cells), and in thesupernatant fluid of cell cultures of cells known to produce angiogenicfactors such as endothelial cells, WI-38 cells (human embryonic lungcells), U937 cells (human lymphoma cells), or SK-Hep (human hepatomacells). Alternatively, the production of these factors may be induced bythe provision of an angiogenesis-stimulating material such as chitosanor a chitosan derivative (Mori et al., 1997, Biomaterials 18:947). Forthis purpose and to provide other invention components, chitosan andchitosan derivatives such as imidazolyl chitosan, methylpyrrolidinonechitosan, etc. are useful at concentrations of 1-90%, preferably 35%-65%of the total composition, as a 1-50% (w/v) chitosan gel, preferably0.5-5%.

The angiogenesis-stimulating nature of a material can be established orconfirmed by various assays known in the art, including theincorporation of the material into a slow release Polymer and implantingthe polymer into a rabbit cornea (Phillips et al., 1997, Wounds 9:1).

Osteoinductive compounds useful in the compositions of the inventioninclude purified materials known to have these characteristics. Suchmaterials alone or in various combinations may include bone morphogenicproteins (1 to n), transforming growth factors (TGF) (1 to n), insulingrowth factors (IGF) (1 to n), platelet derived growth factors (PD-GF),fibroblast growth factors (FGF) (1 to n), tumor necrosis factor (TNF),interleukins (IL) (1 to n), various cytokines, and vitamins such asvitamin D (1-n).

Chitosan has also been shown to promote the differentiation ofmesenchymal stem cells into osteoblasts (Klokkevold et al. 1996 J.Periodontology 67:1170), and may thus serve to stimulate at least a partof the mesenchymalosteoblast differentiation process.

Certain complex materials can also conveniently serve as sources ofosteoinductive molecules. Such molecules are known to be provided bydemineralized bone matrix (“DBM”), which is prepared by grindingcortical bone tissues (generally to 100-500 μm sieved particle size),then treating the ground tissues with hydrochloric acid (generally 0.5to 1 N). See, e.g., Solheim, 1998, Int. Orthop. 22:335-42. DBM iscommercially available, e.g., Grafton® (Osteotech, Eatontown, N.J.);Dynagraft® (GenSci, Irvine, Calif.). It is believed, however, that theacid treatment process used in preparing DBM denatures and/orsolubilizes some of the osteogenic molecules present in untreated bone,destroying the osteogenic nature of the denatured molecules or allowingthem to leach out of the DBM preparation. Therefore, a preferred sourceof osteogenic molecules is non-decalcified bone matrix (“NBM”), which isground cortical bone tissues which are not acid-treated. A combinationof non-decalcified bone matrix protein and DBM is also useful in theinvention compositions as a source of osteoinductive molecules. Theaddition of hyaluronic acid may further enhance the osteoinductivity ofthe mixtures.

The osteoinductive nature of a compound may be determined by knownmethods such as histomorphometric analysis of trabecular bone formationaround a rabbit cranial periosteum implant comprising the putativeosteoinductive compound in DBM. See, e.g., Kibblewhite et al., 1993,Growth Factors 9:185.

The scaffolding materials in the compositions of the present inventionserve to provide direction and a structure for the development of hostneovasculature and osteogenic cells. Materials useful for this purposeinclude hydroxyapatite-chitosan and sulfated-chitosan composites,materials disclosed in U.S. Pat. No. 5,830,493; 5,563,124; 5,755,792; or5,711,957, DBM, or, preferably, cancellous bone, chitosan,chitosan-protein fibers, or chitin-protein fibers. Cancellous bone maybe obtained from any source, including cadavers. When used as ascaffolding material, the cancellous bone is preferably milled to0.1-1.5 mm in its longest diameter. Cancellous bone is used in thesecompositions for its osteoconductive character due to its physicalcharacteristics as a scaffolding material. It is not known to provideany live cells or osteoinductive growth factors. CaSO₄, CaCO₃, and othercalcium salts can also be formed into crystals, either singly orcombined with chitosans, to be used as scaffolding materials. Thescaffolding material is utilized in the compositions at 10-50%,preferably 20-40%.

Chitosan-protein and chitin-protein fibers are preferably produced bythe method described for chitosan-protein fibers in Elgin et al, 1998,Neurological Res. 20:648.

The substance which holds the angiogenesis-stimulating agent andosteogenic molecules and allows the slow release of these components canbe any biocompatible compound known to have these properties. The slowrelease of these factors provides the maximum angiogenic and osteogenicbenefit. See, e.g. Tabata et al., 1998, Biomaterials 19:807. Examples ofmaterials useful for this purpose include gelatin hydrogels (Tabata etal., Id.) and alginate-chitosan (Gaserod et al., 1998, Biomaterials19:1815). As previously discussed, this material, if desired, may alsoprovide a flowable characteristic to the composition. Preferred materialto supply these functions is chitosan gel, which may also serve as amaterial to prevent movement of the composition (see below), as well asan angiogenesis-stimulating agent. Alternatively, theangiogenesis-stimulating agent and/or osteogenic molecules can beencapsulated in microcapsules dispersed in the composition. Themicrocapsules can be composed of, e.g., chitosan or a mixture ofchitosan and alginate (Gaserod et al., Id.), xyloglucan polysaccharidegel (Miyazaki et al., 1998, J. Controlled Release 56:75), or any otherbiocompatible formulation known in the art. The microcapsules may bedispersed evenly throughout the composition. Alternatively, themicrocapsules can be concentrated in an area of the composition wherestimulation of angiogenesis or osteogenesis is most useful. An exampleof such an area is the area adjacent to existing, live bone, where theangiogenesis-stimulating agent would be expected to stimulate theproduction of neovasculature from the live bone.

The invention compositions further comprise a substance for preventingthe composition from moving away from the location where it is placed.This substance is preferably a semi-solid, moldable structure to allowthe composition to be formed into the shape needed for the particularapplication to which the composition is being used. Again, this materialcan also be flowable, angiogenic, osteoconductive, and/orosteoinductive, providing aspects of other components of the inventioncompositions. Useful materials for this purpose include alginate,alginate derivatives, gelatin, hydroxyapatite, tricalcium phosphate,calcium carbonate, and hyaluronic acid. Preferred gel-forming materialsare chitosan and chitosan derivatives such as imidazolyl chitosan ormethylpyrrolidinone chitosan. Combinations of these materials may alsobe utilized advantageously to create the consistency needed for anyparticular application. The component proportions of the materials inthese combinations may be modified to adjust, e.g., the pH, or theconsistency of the composition.

As previously mentioned, certain materials can provide the function ofmore than one component of the invention composition. In particular, theangiogenic/osteogenic slow release component and the component whichprevents the composition from moving can be advantageously combinedwithin one material. Chitosan or chitosan derivatives such as imidazolylchitosan or methylpyrrolidinone chitosan may provide such amulti-component function. The percentage of chitosan is adjusted inthese compositions to provide the desired thickness and flowcharacteristics of the composition, as well as the desired rate ofrelease of the angiogenic and osteogenic growth factors. As previouslydiscussed, chitosan can also wholly or in part provide the angiogenicstimulation component of the compositions.

The thickness of the gels useful for this invention are known to beaffected by other materials present in the composition, particularlycalcium-containing materials such as tricalcium phosphate and calciumchloride. For example, the thickness of chitosan gel is known to beaffected by the presence of tricalcium phosphate. See U.S. Pat. Nos.5,563,124; 5,711,957. Also, the thickness of alginate gels for thisinvention are affected by the presence of calcium chloride as describedin example 1 and shown FIGS. 3A and 3B. Since NBM retains the calciumphosphate from the bone matrix, it provides calcium which can combinewith the chitosan gel to thicken and harden the gel, particularly in anacidic environment. Therefore, the thickness of chitosan gel used in theinvention compositions may be increased by increasing the amount of NBM,or increasing the NBM:DBM ratio, if both materials are used for theosteoinduction component.

Chitosan or chitosan derivatives can also be used alone to stimulatebone growth. In particular the chitosan-based materials can be formedinto solid or tubular structures which can be used as supporting,bridging or guiding structures for bone repair. Chitin-based materialsmay also provide similar functions. These structures can be made, forexample, using the glutaraldehyde cross-linking method disclosed inJameela et al., 1995, Biomaterials 16:769-775. Similar methods can beemployed to make other cross-linked chitin or chitosan materials, e.g.,those cross-linked with carbodiimide, lysine, and vinyl. Incorporatingfibers of these chitin, chitosan or derivatives in multidirectionalwoven or layered patterns provides further strength. For example,solutions of these compounds can be conformed into desired shapes,(e.g., sheets, rods, columns, tubes, etc.) and solidified by, e.g.,drying, curing with vacuum or heat, or addition of salts of minerals(i.e. calcium, sodium). Porous foam-like chitin or chitosan-basedmaterials with a bone-like structure can also be prepared, e.g. by themethod disclosed in Thomson et al., 1995, J. Biomaterials Sci. 7:23-38.These solid materials could also be impregnated with chitosan solutionsbefore or after implantation to fill the structure and add adhesivenessand strength.

The compositions are useful in methods where they are applied to sitesin humans or other vertebrates where bone formation and growth isdesired. Examples of the use of the invention compositions given hereinshould be considered to be non-limiting.

The invention compositions are useful, for example, at sites with bonedefects due to surgery (as occurs, e.g., with the removal of a bonetumor), trauma, or a congenital deficiency (e.g., to correct a cleftpalate). Periodontal applications include the use of the compositions tostrengthen teeth implants and to repair surgically cut facial bones,e.g., mandibles during plastic surgery.

The invention compositions may be applied as a coating of an allograftor autograft used to fill in the defect, or at the junction of the graftand the native bone (the graft-host interface). Alternatively, thecompositions may be applied as a gel or as a rigid, bonelike structure(e.g., comprising a chitosan derivative as previously described),optionally surrounded and/or impregnated with more flowable compositionsto fill in undesired gaps. The rigid structure can serve as a bonereplacement, providing strength and support until new bone replaces thestructure. These rigid structures can, for example, serve as a bonestrut, taking the shape of honeycombed tubular or flat structures. Therigid structures can also be formed into strong hollow tubes to be usedas bridges. For example, the tube can be filled with a chitosan solutionand bridge the two ends of a cylindrical disconnected bone. The rigidstructures are also useful for forming an intervertebral fusion or aspinal prosthesis, e.g., after the removal of a disc, to maintainintervertebral spacing.

A bone can also be replicated in the laboratory using morphometric andbiometric information, e.g., obtained from the original bone. Utilizingvarious imaging techniques such as X-rays, CT and MRI scans, holography,densitometry refraction, etc., the structure, density, configuration,contours, strength, etc. of a particular bone (e.g., a femur head or ametacarpal bone) can be replicated using the invention compositions andinserted as a functional composition. See, e.g., Zentner et al., 1996,Angle Orthodontist 66:463; Ko, 1998, J. Neurosurg. 88:777; Katz et al,1998, Med. Eng. & Physics 20:114; Abe et al., 1998, NeurologiaMedico-Chirurgica 38:746. Components of articulated structures such asartificial knees or hips could also be constructed using thesecompositions. These articulated structures can, for example, compriserigid bone growth-promoting compositions as described herein, cartilageinduced, e.g., by the method disclosed in U.S. Pat. No. 5,837,258, and acapsule, e.g., created by chitosan membranes as disclosed in Chandy etal., 1990, Biomat. Art. Cells, Art. Org. 18:1-24 or Rao et al., 1997, J.Blamed. Mat. Res. 34:21-28. Due to the angiogenic, osteoconductive, andosteoinductive nature of the invention compositions, it would beexpected that the artificial bone structures made from thosecompositions would ultimately be replaced with new host bone.

The composition may also be used at bone fractures to acceleratehealing, or at the junctions between native bone and implants such asknee or hip replacements to prevent loosening of the prosthesis. Inparticular, use of the invention compositions on fractures (e.g. of thevertebra, hip, or wrist) may be indicated for patients withosteoporosis, since the angiogenic and osteogenic properties of thecompositions would be expected to strengthen osteoporotic boneadvantageously.

The composition may be used as a prophylactic treatment to preventfractures in patients with osteoporosis. In this embodiment, bones whichare at risk for fracture in osteoporotic patients are first identifiedby measuring bone density. Bone density is measured using MRI, X-ray,CT-scan, or any other imaging system known in the art for that purpose.The degree of risk for fracture is then assessed based on the bonedensity measurement. The bones with the highest risk for fracture arethen treated with a composition of the present invention by injectingthe composition directly into the bone at points where risk for fractureis highest. A preferred apparatus for performing these injections isthat disclosed in Provisional Patent Application Ser. No. 60/132,852which is herein incorporated by reference. Since the composition mustflow through a cannula and into the bone, the composition to be injectedmust be thinner than compositions which are applied directly to bonedefects. An example of a useful composition for this purpose is NBM,10%; DBM, 10%; cancellous bone, 30%; 2% (w/v) chitosan gel, 50%; 1 mg/mlof a purified vascular endothelial growth factor. Specifically,compositions showing good flow properties may be useful for variousapplications where the use of a syringe or other similar device ispreferred. One example of a useful composition for this purpose is 20%cancellous bone; 20% DBM in 3% alginate. Another example of such acomposition with good flow properties is 30% cancellous bone; 10% DBM in3% alginate. Moreover, another composition with good flow properties is20.6% (w/w) cancellous bone; 12.4% (w/w) DBM; 0.5% (w/w) alginate; 0.3%(w/w) chitosan; 66.2% (w/w) water. An additional composition showinggood flow properties includes 130 mg Hyaluronic acid (HA) solution (1.4%HA solution); 54 mg demineralized bone matrix (DBM); and 130 ng purifiedvascular endothelial growth factor (VEGF). Another composition with goodflow properties is 250 mg of Hyaluronic acid (HA) solution (1.4% sodiumhyaluronate); 105 mg demineralized bone matrix (DBM); and 25 ng purifiedvascular endothelial growth factor (VEGF). Yet another composition withgood flow properties is 125 mg Hyaluronic acid (HA) solution (1.4% HAsolution); 75 mg of crushed cancellous bone; and 125 ng purifiedvascular endothelial growth factor (VEGF).

Various formulations of the invention composition may be prepareddepending on the particular purpose for its application. An example of auseful composition formulation for this invention is a composition whereone or more of the materials is basic fibroblast growth factor, plateletderived endothelial growth factor, or vascular endothelial growth factorpresent at 10⁻⁶ to 30 mg/ml; the demineralized growth factor is presentat 5-30%; the non-decalcified bone matrix is present at 5-30%; thescaffolding material is cancellous bone milled to 0.1-1.5 mm in itslongest diameter and is present at 10-40%; and the gel material is a0.5%-5% (w/v) concentration selected from the group consisting ofchitosan, alginate, hyaluronic acid, a mixture of alginate withchitosan, present at 10-80%, or a mixture of hyaluronic acid andchitosan. For example, when the invention composition is utilized tofill in a large defect, a formulation is utilized which provides arelatively large amount of scaffolding to provide a structure which willsupport the developing vasculature and bone. An example of such aformulation is: cancellous bone, 40-50%, preferably 40%; DBM, 5-30%,preferably 10%; NBM, 5-30%, preferably 10%; 1 mg/ml of a purifiedvascular endothelial growth factor; 5% (w/v) chitosan gel, 20-50%,preferably 40%. Another example of a relatively friable composition is50% (v/v) cancellous bone; 10% (v/v) DBM in 3% alginate.

If the invention composition is utilized to stimulate bone formationwhere a supporting scaffolding is not needed, such as at the site of afracture, or around an allograft or autograft, a minimal amount ofscaffolding is utilized, but a relatively large amount of angiogenicfactors may be advantageous to promote rapid fusion at the fracture orgraft-host interface. A relatively large amount of gel-forming materialmay also be advantageous in this situation to assure minimal movement ofthe composition. An example of such a formulation is: cancellous bone,0-30%, preferably 10%; DBM, 5-15%, preferably 10%; NBM, 5-15%,preferably 10%; up to 10 mg/ml of a purified vascular endothelial growthfactor; 3-10% (w/v) chitosan gel, present at 20-90% of the composition,preferably a 5% gel, present at 70%.

Additionally, other methods to insure the minimal amount of movement ofthe composition from the site of application may be used. For example,the topical application of calcium to an alginate excipient causes thecomposition to remain in place, and reduces the flow properties of thecomposition. The experimental data described below demonstrates theretention properties of a composition using an alginate excipient withthe addition of calcium after the composition has been localized to thesite of the bone defect. FIG. 3A illustrates the localized appearance ofbone allograft material contained in alginate at one weekpost-implantation when there was no addition of calcium to the alginatemixture. The allograft was observed to migrate from the defect into thehip joint. In contrast, FIG. 3B shows the localized appearance of boneallograft material contained in alginate at one week post-implantationwhere there was an addition of calcium to the alginate mixtureimmediately after it was placed in the site of bone defect.

The following examples are intended to illustrate but not to limit thepresent invention. In light of the detailed description of the inventionand the examples presented below, it can be appreciated that the severalaspects of the invention are achieved. It is to be understood that thepresent invention has been described in detail by way of illustrationand example in order to acquaint others skilled in the art with theinvention, its principles, and its practical application. Particularformulations and processes of the present invention are not limited tothe descriptions of specific embodiments presented, but rather thedescriptions and examples should be in terms of the claims that followand their equivalents. While some of the examples and descriptions belowinclude some conclusions about the way the invention may function, theinventor does not intend to be bound by those conclusions and functions,but puts them forth only as possible explanations.

EXAMPLE 1 Surgical Model

This procedure briefly describes the surgical model employed to evaluatethe healing potential of bone allograft material in an alginate carrier,following implantation within a clinically significant bone defect. Thetechnique was reproduced from the following reference: Bruder et al.,“Bone Regeneration by Implantation of Purified, Culture Expanded HumanMesenchymal Stem Cells,” J. Orthopaedic Research 16:155162 (1998).

A bilateral surgical model was created to evaluate a novel compositionof human allograft bone in an excipient carrier when placed in aclinically significant osseous defect. Such defects fail to undergointrinsic repair without osteogenic/osteoinductive augmentation.

Both femurs of athymic nude rats 12-16 weeks of age (National CancerInstitute, Rnu −/−) were exposed via an anterolateral surgical approachas shown in FIGS. 1A and 1B. A predrilled high density polyethylenefixation plate measuring 4 mm×4 mm×23 mm, with 9 mm pre-drilled centerholes (straight) was subsequently attached to each femur by four (4)Kirschner wires and two (2) cerclage wires as shown in FIG. 1C. A 5 mmtransverse segment of the central diaphysis, including the adherentperiosteum, was removed using a side cutting burr under salineirrigation as shown in FIG. 2B. Excipients used in the current studiesconsisted either of 3% alginate from various sources or high viscosityhyaluronic acid (Healon G V, Pharmacia/Upjohn). Bone allograft materialwas held in place via the apposing musculature, and the fascia and skinwere closed using 5-0 Ethibond and 5-0 Ethilon sutures, respectively.Animals were euthanized between 1 and 12 weeks for radiographicevaluation of new bone formation. Femurs were collected and fixed in 10%neutral buffered formalin and processed for histologic evaluation uponstaining with either saffranin-O/fast green or geimsa.

Results:

FIG. 5 illustrates the appearance of bone formation at the site of bonedefect after the application of the composition containing demineralizedbone matrix(DBM), Hyaluronic acid (HA), and purified vascularendothelial growth factor (VEGF) at 12 weeks. The composition used forthe animal of FIG. 5 is 130 mg of HA solution (1.4% sodium hyaluronate),54 mg of DBM, and 13 Ong of VEGF. FIG. 6 is a histologic evaluation ofthe new bone formation shown in FIG. 5. In FIG. 6 the pink areademonstrates excellent new bone formation.

Comparison of Negative Controls During Model Development

FIG. 4 shows a comparison of the results obtained at 12 weekspostoperatively. Panels A and C represent negative controls in whichfemoral defects were either left untreated or filled with alginateexcipient alone, respectively. Note that there is no bridging of thedefect with new bone.

Calcium Augmentation of Alginate Excipient Prevents Migration of BoneAllograft Material

Alginate was dissolved in PBS (final concentration 3%) and mixed withbone allograft prior to implantation as described above. Once thedefects were filled, calcium chloride solution (50 μl, 5 mM) was appliedtopically to the alginate/bone material, at which point the excipientmaterial polymerized to form a hardened gel. Identical material in thecontralateral limb was left untreated. Animals were sacrificed at 1 and2 weeks for radiographic evaluation of the implanted bone material, andthe tissues were subsequently harvested for histologic evaluation. FIG.3A illustrates the localized appearance of bone allograft material inalginate without the addition of calcium one (1) week post implantation.The allograft was observed to migrate from the defect into the hip jointwhere heterotopic ossification occurred within a 12 week period. Incontrast, topical application of calcium to the alginate excipientcaused the bone allograft to be retained within the defect as shown inFIG. 3B.

Other features, objects and advantages of the present invention will beapparent to those skilled in the art. The explanations and illustrationspresented herein are intended to acquaint others skilled in the art withthe invention, its principles, and its practical application. Thoseskilled in the art may adapt and apply the invention in its numerousforms, as may be best suited to the requirements of a particular use.Accordingly, the specific embodiments of the present invention as setforth are not intended as being exhaustive or limiting of the invention.

What is claimed is:
 1. A composition for promoting growth andstrengthening of bone comprising a mixture of a chitosan or chitosanderivative, cancellous bone, and demineralized bone matrix; wherein thechitosan derivative is imidazolyl chitosan or methylpyrrolidoninonechitosan.
 2. A composition of claim 1, wherein the demineralized bonematrix is present at 10%; the cancellous bone is present at 10%; and thecomposition comprises a gel material constituted 3% (w/v) of thechitosan or the chitosan derivative.
 3. A composition of claim 1,wherein the demineralized bone matrix is present at 20%; the cancellousbone is present at 20%; and the composition comprises a gel materialconstituted 3% (w/v) of the chitosan or the chitosan derivative.
 4. Acomposition of claim 1, wherein the demineralized bone matrix is presentat 10%; the cancellous bone is present at 30%; and the compositioncomprises a gel material constituted 3% (w/v) of the chitosan or thechitosan derivative.
 5. A method of inducing bone formation in avertebrate comprising applying a composition of claim 1 to a site in thevertebrate where bone formation is desired.
 6. The method of claim 5,wherein the site is the junction of an allograft or autograft and abone.
 7. The method of claim 5, wherein the site is the junction of abone and a bone prosthesis.
 8. The method of claim 5, wherein the siteis a fracture.
 9. A composition for promoting growth and strengtheningof bone comprising a mixture of alginate, calcium, cancellous bone, anddemineralized bone matrix.
 10. A composition of claim 9, wherein thedemineralized bone matrix is present at 10%; the cancellous bone ispresent at 30%; and the composition comprises a gel material constituted3% (w/v) of the alginate.
 11. A composition of claim 9, wherein thedemineralized bone matrix is present at 20%; the cancellous bone ispresent at 20%; and the composition comprises a gel material constituted3% (w/v) of the alginate.
 12. A composition of claim 9, wherein thedemineralized bone matrix is present at 10%; the cancellous bone ispresent at 10%; and the composition comprises a gel material constituted3% (w/v) of the alginate.
 13. A composition, comprising: (a) one or moreangiogenesis-stimulating materials; (b) an osteoinductive material; (c)a scaffolding material comprising cancellous bone; and (d) a gelmaterial.
 14. The composition of claim 13 wherein the gel materialcomprises alginate.
 15. The composition of claim 14, also comprisingcalcium chloride or tricalcium phosphate.
 16. The composition of claim13 wherein the gel material comprises chitosan, imidazolyl chitosan ormethylpyrrolidoninone chitosan.
 17. The composition of claim 13, whereinthe osteoinductive material comprises demineralized bone matrix.
 18. Thecomposition of claim 13, wherein the osteoinductive material comprises abone morphogenic protein.